Disassembly of Escherichia coli RecA E38K/DeltaC17 nucleoprotein filaments is required to complete DNA strand exchange.

Disassembly of RecA protein subunits from a RecA filament has long been known to occur during DNA strand exchange, although its importance to this process has been controversial. An Escherichia coli RecA E38K/DeltaC17 double mutant protein displays a unique and pH-dependent mutational separation of DNA pairing and extended DNA strand exchange. Single strand DNA-dependent ATP hydrolysis is catalyzed by this mutant protein nearly normally from pH 6 to 8.

An SOS inhibitor that binds to free RecA protein: the PsiB protein.

The process of bacterial conjugation involves the transfer of a conjugative plasmid as a single strand. The potentially deleterious SOS response, which is normally triggered by the appearance of single-stranded DNA, is suppressed in the recipient cell by a conjugative plasmid system centered on the product of the psiB gene. The F plasmid PsiB protein inhibits all activities of the RecA protein, including DNA binding, DNA strand exchange, and LexA protein cleavage.

DdrB protein, an alternative Deinococcus radiodurans SSB induced by ionizing radiation.

Deinococcus radiodurans exhibits an extraordinary resistance to the effects of exposure to ionizing radiation (IR). DdrB is one of five proteins induced to high levels in Deinococcus following extreme IR exposure and that play a demonstrable role in genome reconstitution. Although homology is limited, DdrB is a bacterial single-stranded DNA-binding protein.

RecA-mediated SOS induction requires an extended filament conformation but no ATP hydrolysis.

The Escherichia coli SOS response to DNA damage is modulated by the RecA protein, a recombinase that forms an extended filament on single-stranded DNA and hydrolyzes ATP. The RecA K72R (recA2201) mutation eliminates the ATPase activity of RecA protein. The mutation also limits the capacity of RecA to form long filaments in the presence of ATP.

Defective dissociation of a "slow" RecA mutant protein imparts an Escherichia coli growth defect.

The RecA and some related proteins possess a simple motif, called (KR)X(KR), that (in RecA) consists of two lysine residues at positions 248 and 250 at the subunit-subunit interface. This study and previous work implicate this RecA motif in the following: (a) catalyzing ATP hydrolysis in trans,(b) coordinating the ATP hydrolytic cycles of adjacent subunits, (c) governing the rate of ATP hydrolysis, and (d) coupling the ATP hydrolysis to work (in this case DNA strand exchange). The conservative K250R mutation leaves RecA nucleoprotein filament formation largely intact.

Microplitis demolitor bracovirus genome segments vary in abundance and are individually packaged in virions.

Polydnaviruses (PDVs) are distinguished by their unique association with parasitoid wasps and their segmented, double-stranded (ds) DNA genomes that are non-equimolar in abundance. Relatively little is actually known, however, about genome packaging or segment abundance of these viruses. Here, we conducted electron microscopy (EM) and real-time polymerase chain reaction (PCR) studies to characterize packaging and segment abundance of Microplitis demolitor bracovirus (MdBV).

Thermus thermophilus bacteriophage phiYS40 genome and proteomic characterization of virions.

We determined the sequence of the 152,372 bp genome of phiYS40, a lytic tailed bacteriophage of Thermus thermophilus. The genome contains 170 putative open reading frames and three tRNA genes. Functions for 25% of phiYS40 gene products were predicted on the basis of similarity to proteins of known function from diverse phages and bacteria.

Complementation of one RecA protein point mutation by another. Evidence for trans catalysis of ATP hydrolysis.

The RecA residues Lys248 and Glu96 are closely opposed across the RecA subunit-subunit interface in some recent models of the RecA nucleoprotein filament. The K248R and E96D single mutant proteins of the Escherichia coli RecA protein each bind to DNA and form nucleoprotein filaments but do not hydrolyze ATP or dATP. A mixture of K248R and E96D single mutant proteins restores dATP hydrolysis to 25% of the wild type rate, with maximum restoration seen when the proteins are present in a 1:1 ratio.

The RecF protein antagonizes RecX function via direct interaction.

The RecX protein inhibits RecA filament extension, leading to net filament disassembly. The RecF protein physically interacts with the RecX protein and protects RecA from the inhibitory effects of RecX. In vitro, efficient RecA filament formation onto single-stranded DNA binding protein (SSB)-coated circular single-stranded DNA (ssDNA) in the presence of RecX occurs only when all of the RecFOR proteins are present.

Inhibition of RecA protein function by the RdgC protein from Escherichia coli.

The Escherichia coli RdgC protein is a potential negative regulator of RecA function. RdgC inhibits RecA protein-promoted DNA strand exchange, ATPase activity, and RecA-dependent LexA cleavage. The primary mechanism of RdgC inhibition appears to involve a simple competition for DNA binding sites, especially on duplex DNA.

Genome sequence and gene expression of Bacillus anthracis bacteriophage Fah.

Fah, a lytic bacteriophage of Bacillus anthracis, is used widely in the former Soviet Union to identify anthrax bacteria. Here, we present the analysis of a 37,974 bp sequence of the Fah genome and examine gene expression of the phage in a model host, Bacillus cereus. Half of the Fah genome contains genes coding for structural proteins and host lysis functions in an arrangement typical of Syphoviridae.

Role of pi dimers in coupling ("handcuffing") of plasmid R6K's gamma ori iterons.

One proposed mechanism of replication inhibition in iteron-containing plasmids (ICPs) is "handcuffing," in which the coupling of origins via iteron-bound replication initiator (Rep) protein turns off origin function. In minimal R6K replicons, copy number control requires the interaction of plasmid-encoded pi protein with the seven 22-bp iterons of the gamma origin of replication. Like other related Rep proteins, pi exists as both monomers and dimers.

A RecA filament capping mechanism for RecX protein.

The RecX protein is a potent inhibitor of RecA protein activities. RecX functions by specifically blocking the extension of RecA filaments. In vitro, this leads to a net disassembly of RecA protein from circular single-stranded DNA.

The DinI protein stabilizes RecA protein filaments.

When DinI is present at concentrations that are stoichiometric with those of RecA or somewhat greater, DinI has a substantial stabilizing effect on RecA filaments bound to DNA. Exchange of RecA between free and bound forms was almost entirely suppressed, and highly stable filaments were documented with several different experimental methods. DinI-mediated stabilization did not affect RecA-mediated ATP hydrolysis and LexA co-protease activities.

Situational repair of replication forks: roles of RecG and RecA proteins.

Replication forks often stall or collapse when they encounter a DNA lesion. Fork regression is part of several major paths to the repair of stalled forks, allowing nonmutagenic bypass of the lesion. We have shown previously that Escherichia coli RecA protein can promote extensive regression of a forked DNA substrate that mimics a possible structure of a replication fork stalled at a leading strand lesion.

Genome and proteome of Listeria monocytogenes phage PSA: an unusual case for programmed + 1 translational frameshifting in structural protein synthesis.

PSA is a temperate phage isolated from Listeria monocytogenes strain Scott A. We report its complete nucleotide sequence, which consists of a linear 37 618 bp DNA featuring invariable, 3'-protruding single stranded (cohesive) ends of 10 nucleotides. The physical characteristics were confirmed by partial denaturation mapping and electron microscopy of DNA molecules.

Genome of Xanthomonas oryzae bacteriophage Xp10: an odd T-odd phage.

Xp10 is a lytic bacteriophage of the phytopathogenic bacterium Xanthomonas oryzae. Though morphologically Xp10 belongs to the Syphoviridae family, it encodes its own single-subunit RNA polymerase characteristic of T7-like phages of the Podoviridae family. Here, we report the determination and analysis of the 44,373 bp sequence of the Xp10 genome.

C-terminal deletions of the Escherichia coli RecA protein. Characterization of in vivo and in vitro effects.

A set of C-terminal deletion mutants of the RecA protein of Escherichia coli, progressively removing 6, 13, 17, and 25 amino acid residues, has been generated, expressed, and purified. In vivo, the deletion of 13 to 17 C-terminal residues results in increased sensitivity to mitomycin C. In vitro, the deletions enhance binding to duplex DNA as previously observed.

Circadian clock protein KaiC forms ATP-dependent hexameric rings and binds DNA.

KaiC from Synechococcus elongatus PCC 7942 (KaiC) is an essential circadian clock protein in cyanobacteria. Previous sequence analyses suggested its inclusion in the RecADnaB superfamily. A characteristic of the proteins of this superfamily is that they form homohexameric complexes that bind DNA.

The Rad51-dependent pairing of long DNA substrates is stabilized by replication protein A.

Rad51 protein forms nucleoprotein filaments on single-stranded DNA (ssDNA) and then pairs that DNA with the complementary strand of incoming duplex DNA. In apparent contrast with published results, we demonstrate that Rad51 protein promotes an extensive pairing of long homologous DNAs in the absence of replication protein A. This pairing exists only within the Rad51 filament; it was previously undetected because it is lost upon deproteinization.

RecA Protein from the extremely radioresistant bacterium Deinococcus radiodurans: expression, purification, and characterization.

The RecA protein of Deinococcus radiodurans (RecA(Dr)) is essential for the extreme radiation resistance of this organism. The RecA(Dr) protein has been cloned and expressed in Escherichia coli and purified from this host. In some respects, the RecA(Dr) protein and the E.